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a Dep. of Plant and Soil Sci., Mississippi State Univ., Mississippi State, MS 39762
b Crop and Soil Environ. Sci. Dep., Virginia Polytechnic Institute and State Univ., Blacksburg, VA 24061
c Dep. of Agric. Sci., Texas A&M Univ.-Commerce, Commerce, TX 75429
* Corresponding author (gmunshaw{at}pss.msstate.edu)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviations: CER, CO2 exchange rate • SWE, seaweed extract • TNC, total nonstructural carbohydrate
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Although there are conflicting reports (Schmidt and Blaser, 1969; Beard, 1973) regarding the effect of fall fertilization of bermudagrass, recent research suggests that earlier reports of negative effects of late-season N applications on bermudagrass cold tolerance may not be accurate. Goatley et al. (1994) found that late-season N improved fall and spring color and had little effect on total nonstructural carbohydrate (TNC) levels. Schmidt and Chalmers (1993) reported that the positive effects of late-season N applications (better color, longer color retention) occurred without any negative effects on post dormancy recovery in the spring.
Richardson (2001) found that late-season N fertility had no influence on seeded bermudagrass winter survival. Richardson (2002) also found that late-season N applications on Tifway bermudagrass increased fall color, enhanced spring greenup, and had no effect on rhizome cold tolerance. Although many studies have examined the effects of late-season N on TNC concentrations, virtually no work has examined the influence on other physiological parameters.
Iron may be another nutrient that prolongs fall color without negatively affecting cold tolerance. Tests have shown that Fe applications in conjunction with moderate (24 kg ha–1 mo–1) summer N applications can improve the performance of bermudagrass during the fall and improve recovery in the spring (White and Schmidt, 1990). White and Schmidt (1989) also report that Fe maintained the aesthetic quality of cold-sensitive and cold-tolerant bermudagrass cultivars after a chilling period and assisted in CO2 exchange rate (CER) recovery.
Certain natural products such as seaweed (Ascophyllum nodosum Jol.) extract contain high levels of cytokinins and auxins as well as moderate levels of other hormones (Mooney and Van Staden, 1986; Crouch et al., 1992). Verkleij (1992) stated that the efficacy of these products appeared to be due mainly to cytokinins but may also have been due to trace nutrients found in the products. A recent study by Zhang and Ervin (2004) with creeping bentgrass (Agrostis stolonifera L.) also indicates that beneficial effects of SWE applications such as increased levels of antioxidants and photochemical efficiency during drought may be due to increased endogenous levels of cytokinins.
Cytokinins can act to inhibit senescence in leaves by counteracting the effects of ethylene or abscisic acid (Arteca, 1996; Buchanan et al., 2000). Cytokinins may also maintain membrane integrity by reducing lipase and lipoxygenase activity, processes involved in membrane breakdown (Mok, 1994). Goatley and Schmidt (1990) reported antisenescence responses in excised Kentucky bluegrass (Poa pratensis L.) leaves after treatment with the synthetic cytokinin benzyladenine. However, White and Schmidt (1990) found that treating bermudagrass with benzyladenine did not consistently affect fall color or quality and did not influence carbohydrate levels. Similarly, Nakamae and Nakamura (1982) did not see an increase in leaf chlorophyll content of Manila grass [Zoysia matrella (L.) Merr. var. matrella] during autumn after applications of 6-benzyladenine.
Plant membranes must be kept in a fluid state to maintain function. When membranes become less fluid or gel-like, their protein components become impaired or nonfunctional (Samala et al., 1998; Taiz and Zeiger, 1998). Plants maintain membrane fluidity at lower temperatures by increasing the unsaturation of lipids in the phospholipid bilayer. Studies examining fatty acids in C4 grasses have shown that during cold acclimation, there is an increase in the unsaturated/saturated ratio (Samala et al., 1998; Cyril et al., 2001, 2002). This increase occurred to a greater extent in cold-tolerant cultivars than cold-sensitive cultivars.
During cold stress, plants accumulate sucrose and other simple sugars as well as proline and glycine betaine, which have been reported to help stabilize membranes and act as osmolytes that maintain water balance within the cell (Nilsen and Orcutt, 1996; Holmstrom et al., 2000). Proline may have many functions in stress tolerance, including osmotic adjustment, protein and membrane stabilization, gene induction, reactive oxygen species scavenging, N and C source, and a reduction equivalent source during stress recovery (Rudolph et al., 1986; Delauney and Verma, 1993; Saradhi et al., 1995; Hare and Cress, 1997; Iyer and Caplan, 1998; Brugiere et al., 1999). Proline levels have been shown to increase during cold acclimation, decrease during de-acclimation, and increase to a greater extent in cold-hardy species (Levitt, 1980). As extracellular ice crystals form, water moves from inside the cell to enlarge these extracellular ice crystals. By increasing solute concentration, cell osmotic potential is decreased, making water movement out of the cell less likely. This reduction would prevent the enlargement of ice crystals and maintain cell hydration (Rossi, 1997).
This study was designed to examine the effects of late-season N, Fe, and SWE applications on fall through spring aesthetic responses, fatty acid saturation levels, and proline concentrations in the stolon tissues of four bermudagrass cultivars. An understanding of these factors will help to better define the effects that late-season nutrient applications have on physiological processes occurring during acclimation and de-acclimation. The objectives of this study were to (i) determine the effects of late-season N, Fe, and SWE applications on bermudagrass fall visual quality, spring greenup, lipid saturation, and proline concentration; (ii) determine if these treatments were associated with changes in freezing tolerance; and (iii) determine biochemical and cold tolerance differences in four bermudagrass cultivars.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Seeded plots were planted under a geotextile fabric to discourage seed movement as well as enhance germination and plant development. Plots were mowed three times per week with a reel mower set at 1.91 cm. Nitrogen was applied in the form of NH4NO3 (34–0–0) once monthly at a rate of 48.8 kg N ha –1 beginning at establishment and ending 15 August. A complete fertilizer (10N–4.4P–8.3K) was applied the following spring (25 May) at a rate of 48.8 kg N ha–1, and monthly N applications (from 34–0–0) began again on 15 June 2002. Irrigation was supplied as needed to prevent visual moisture stress.
After bermudagrass establishment, three chemical treatments were applied during the summer to autumn period leading to bermudagrass shoot senescence. A nontreated control was also included within each bermudagrass cultivar.
Fall Chemical Treatments
Fall chemical treatments began 15 August in 2001 and 2002 and continued on a 3-wk schedule until apparent dormancy (80–100% canopy browning). Final chemical treatment dates were 17 Oct. 2001 and 31 Oct. 2002. Seaweed extract was applied at a rate of 0.54 kg ha–1 (Zhang et al., 2002); N was applied at a rate of 48.8 kg N ha–1 as NH4NO3 (White and Schmidt, 1990); and Fe was applied at a rate of 1 kg Fe ha–1 as FeSO4 (Schmidt and Chalmers, 1993). Chemical subplots measured 3.1 by 1.8 m.
The experimental design was a split plot arrangement of treatments in a randomized complete block with repeated measures and four replications. Cultivar was considered whole plot factor, while chemical treatment was considered subplot factor. Data were analyzed using the mixed procedure of the Statistical Analysis System (SAS) software (SAS, Cary, NC) (SAS, 2003). Appropriate main effects and interactions were separated using Fisher's Protected LSD test at an 
level of 0.05. The study was conducted during the 2001 through 2002 growing season and repeated during the 2002 through 2003 growing season. This design holds true for all response variables with the exception of controlled freezing.
Fall Quality and Spring Greenup
Visual turfgrass quality ratings were taken (using a 1 to 9 scale, where 1 = completely brown, dormant or dead turf, and 9 = lush green turf) monthly during autumn. As plots were well established by late autumn, color was the primary parameter of interest during quality ratings. Spring greenup was visually estimated as the percentage of green ground cover present.
Controlled Freezing
Freeze chamber analyses were performed on acclimating (fall) and acclimated (winter) stolon tissues in 2001–2002 and 2002–2003. Additional analysis was conducted on samples collected in summer 2003. Control samples only were collected during the acclimating period of both years. This was done as the freezing process is time consuming and the possibility of physiological differences occurring in samples between the first and last tests of each sampling period existed. Samples were analyzed from all plots in winter each year and in summer 2003. The experimental design was a split-split plot arrangement of treatments in a randomized complete block with repeated measures and four replications. Cultivar was considered whole plot factor, chemical treatment was considered subplot factor, and temperature regime was considered sub-subplot factor. Data were analyzed using the mixed procedure of SAS (SAS, 2003). Appropriate main effects and interactions were separated using Fisher's Protected LSD test at an level of 0.05. A 10.2-cm (diameter) cup cutter sample was removed from each plot, cleaned of soil by washing, and divided into four equal subsamples. One of the subsamples was placed in a refrigerator and held at 4.0°C to act as a "control." The other three subsamples were placed in a freeze chamber that was programmed to ramp from 8.0 to 1.0°C overnight. The temperature then ramped to –2.8°C over a 2-h period, stayed at this temperature for 0.5 h, and a subsample was removed. This process continued with ramping to –5.0 and then –7.2°C over the next 5 h. After removal, subsamples were held over night at 4.0°C and then placed in a sand-filled mist bench in the glasshouse at 22 ± 2°C. Temperatures were verified with a Watchdog 400 data logger (Spectrum Technologies, Inc., Plainfield, IL). Regrowth was visually estimated as the percentage of the sample exhibiting shoot regrowth or appearing green approximately 4 wk after freezing (Schmidt and Chalmers, 1993).
Total Lipid Extraction and Fatty Acid Quantification
Tissue samples were removed from the field during acclimation (November), when acclimated (January–February), and when nonacclimated (July) and stored at –80°C until analyzed. For each analysis, 1 g of stolon tissue was ground with a mortar and pestle in liquid N2. This ground tissue was then transferred to a centrifuge tube for total lipid extraction using 3 mL of a buffer containing chloroform/methanol/water (1:2:0.8). After soaking for 1 h at room temperature, 1 mL 1% NaCl and 3 mL chloroform were added and centrifugation occurred at 1200 g for 10 min. The lower chloroform layer containing the lipids was transferred to a test tube, and the chloroform addition, centrifugation, and chloroform layer transfer were repeated two more times (Cyril et al., 2001).
In a method described by Goyal (2000) and modified by Shang et al. (2005), the chloroform layer was then evaporated under a stream of N2. After evaporation, 5 mL of 2% NaOH in 90% methanol was added, and tubes were placed in a water bath at 75 to 80°C for 30 min. Tubes had an air-cool reflux (small funnels placed in tubes) during this process to facilitate hydrolysis. At the end of 30 min, the mouths of the tubes were left open to allow for evaporation of the methanol under a fume hood. Next, 2 mL of distilled–deionized (DD) H2O were added to facilitate dissolution, and the contents were transferred to a 10-mL screw-cap tube. The residue was washed twice more with 2 mL DD H2O, and contents were transferred to a new tube. To this new tube was added 300 µL of 6 M H2SO4 to precipitate Na salts out of the fatty acids. The acid form of the fatty acids was recovered by adding 1 mL hexane and centrifuging at 150 g for 5 min. After centrifugation, the hexane layer containing the free fatty acids was transferred to a new 10-mL screw-cap tube and the process was repeated two more times. After the final centrifugation, the hexane volume was reduced to 100 µL under a gentle stream of N2 gas, and 100 µL -bromoacetephenone (10 mg mL–1 acetone) and 100 µL triethylamine (TEA) (10 mg mL–1 acetone) were added and caps were tightly fastened. Tubes were placed into a water bath at 100°C for 15 min. Free fatty acids react with -bromoacetophenone in the presence of TEA and produce a derivative that is UV sensitive and can be quantified with a UV detector after HPLC separation. Tubes were then allowed to cool, and 140 µL acetic acid (2 mg mL–1 acetone) was added and the tubes were placed back in the 100°C water bath for 5 min. After cooling a second time, the content was dried under a stream of N2 to inactivate the remaining reagents. The residue was dissolved with 500 µL acetonitrile, and the solution was filtered with a 0.2-µm membrane before injection into HPLC.
HPLC Procedures
Chromatographic analyses were performed on an Agilent (Agilent Technologies, Palo Alto, CA) 1100 series HPLC system with a photodiode array detector. An Ultrasphere-C8 (250 by 4.6 mm, 5 µm) (Beckman Coulter, Inc., Fullerton, CA) analytical column with a C-8 guard column (7.5 by 4.6 mm) (Alltech Associates, Deerfield, IL) was used for chromatographic separation. The mobile phase was 90% acetonitrile in water. Samples were eluted at a gradient rate: from 1 mL min–1 up to 2 mL min–1 within the first 2 min, at an isocratic elution of 2 mL min–1 for 10 min, and then down to 1 mL min–1 within the next 8 min. Total elution time per sample was 20 min. The injection volume was 20 µL. The fatty acid derivatives were quantified at a wavelength of 214 nm. The retention times (minutes) at the described conditions were 4.6, 5.8, 7.3 and 10.8 for linolenic, linoleic, palmitic and stearic acids, respectively. The limit of identification for the above procedure is 0.07 to 0.6 µmol per injection using three times the standard deviation for one-half of the lowest standard.
Fatty acid standards were purchased from Sigma Chemical Co. (Sigma-Aldrich, St. Louis, MO). External standards prepared from the commercial standards were used for calibration. Two reference samples, which were preweighed from a composite tissue sample, were included in each analysis set.
Proline Determination
Additional stolon samples were removed at the same time as the controlled freezing tests (fall, winter, and summer), placed in liquid N2 to halt respiration and then placed in a –80°C freezer for later analysis. Stolons were ground with a mortar and pestle in liquid N2 and approximately 0.20 g stolon material was homogenized in 10 mL of 3% sulfosalicylic acid, and the homogenate was filtered through Whatman no. 2 filter paper. Two milliliters of acid ninhydrin and 2 mL glacial acetic acid were added to 2 mL of the filtrate and incubated at 100°C for 1 h. The reaction was terminated by placing test tubes in an ice bath. The mixture was extracted with 4 mL toluene and vortexed for 15 to 20 s. The chromophore containing toluene was warmed to room temperature and absorbance read at 520 nm using toluene as the blank. Proline concentration was determined from a standard curve (Syvertsen and Smith, 1983).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Iron did not significantly improve fall color during the evaluation period. White and Schmidt (1990) did not observe any differences with the application of Fe until air temperatures reached freezing. Schmidt and Chalmers (1993) found a 20% improvement in October turfgrass color due to Fe, but only when no N was applied in September. Rodgers (2003) suggests that the new seeded bermudagrasses use Fe more efficiently than older seeded cultivars. Although no older seeded cultivars were examined in this study, the interaction between cultivar and chemical treatment was not observed. The effect of Fe was minimal on all cultivars. A higher rate of FeSO4 or a chelated form of Fe may have resulted in a greater response through the fall due to a greater chance of Fe uptake.
Seaweed extract applications did not have any effect on bermudagrass color retention, as quality ratings were very similar to the control in both years of the study. This finding is in agreement with White and Schmidt (1990), who found that synthetic cytokinin applications did not have a consistent effect on late-season bermudagrass quality or color. Seaweed extracts also contain cytokinins and the response on bermudagrass appears to be very similar with the two products.
Controlled Freezing
As the freeze chamber temperature decreased from 4.0 to –7.2°C, bermudagrass survival generally decreased (Tables 3 and 4). Further, at all winter sampling dates, Midiron typically had the highest amount of survival and regrowth following freezing, especially following exposure to the colder temperatures. Riviera normally had the next highest amount of survival, followed by Tifway and finally Princess-77. These results are in agreement with previous work where Midiron was described as cold tolerant. Midiron is followed by Riviera with moderate cold tolerance, Tifway somewhat cold susceptible, and Princess-77 having poor cold tolerance (Shashikumar and Nus, 1993; Anderson et al., 2003).
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In 2002, the first killing frost did not occur until 2 November, and temperatures generally remained cool until the fall sampling date. This 3-wk period of temperatures dropping below freezing may have been enough to harden samples to a level not attained in 2001. The amount of survival after freezing to –5.0 and –7.2°C in fall 2002 was much higher than in fall 2001. This indicates that the combination of the 3-wk period in early November and a gradual decline in temperatures throughout the fall were sufficient for acclimation. Gatschet et al. (1994) found that lethal temperatures were lowered 5°C by acclimating bermudagrass for 4 wk at 8/2°C day/night temperatures. The 3-wk period in November had a mean temperature of 6.7°C, with a high of 20 and many lows below 0°C.
Survival at lower temperatures was generally greater in winter than in fall of each year (Table 3 and 4). Clearly, acclimation was still occurring after the fall sampling dates. Anderson and Taliaferro (1995) subjected bermudagrass to freezing temperatures at monthly intervals throughout the fall and winter and found survival was higher in midwinter than in November. There were generally no differences in survival between cultivars in winter 2002 and 2003 (Table 4). Air temperatures leading up to the winter 2002 sampling date had reached as low as –14.4°C, which may have caused regrowth after 4.0°C to be less than 100%. Temperatures leading up to the winter 2003 sampling date had reached as low as –16.9°C, but due to the insulating effects of snow cover, soil temperatures (data not shown) were actually warmer in 2003 than in 2002, allowing for similar amounts of regrowth for both years.
Sampling stolons from the field in summer 2003 and storage at 4.0°C indicated that chilling temperatures had differing effects on survival of unacclimated samples, with Riviera having more regrowth than Midiron (Table 4). Although seeded bermudagrasses are normally slow to establish, subsequent years after sowing show much greater stolon and rhizome development. This increased density in the summer may have allowed Riviera to become more efficient photosynthetically and to allow the storage of more TNC. Hensler et al. (1999) suggested and Munshaw et al. (2001) reported that increased stolon diameter results in higher TNC levels. Although stolon diameters and TNC were not measured in the current study, they may play an important role in freezing survival. None of the summer 2003 sampled cultivars survived even the most moderate of freezing temperatures. Anderson et al. (1988) found that freeze tests conducted on bermudagrass cultivars in June resulted in much less survival than during the winter.
The effects of sampling date, cultivar, and fall chemical treatment interacted with respect to postfreeze regrowth (Table 5). As explained above, fall treatment effects were not tested in November of either year. In general, there was little effect of chemical treatment on bermudagrass survival across all temperatures and sampling dates, and no chemical treatment consistently reduced survival relative to the control. Turfgrass managers growing bermudagrass typically discontinue N use in late summer with the belief that late-season–applied N will increase succulence and winter injury. Controlled freezing tests on cold-acclimated bermudagrass samples in this study indicated that, across all temperatures, N treatment did not affect freezing tolerance of any cultivar. Richardson (2002) froze late-season N-treated bermudagrass rhizomes to similar temperatures and also did not find any negative effects on freezing tolerance. Further, Schmidt and Chalmers (1993) evaluated regrowth after controlled freezing and did not find any negative effects of late-season N applications.
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Schmidt and Chalmers (1993) observed improved bermudagrass postfreeze regrowth following applications of SWE fortified with humic acid and thiamine. In the present study, SWE alone did not influence postfreeze regrowth (with the exception of Tifway in winter 2002 [=0.10]), indicating that the positive results seen by Schmidt and Chalmers may have been due to humic acid, thiamine, or interactions of the three compounds.
Fatty Acid Analysis
The absolute amounts of key fatty acids in total polar lipids (mainly phospholipids) for each cultivar are shown in Table 6. With the exception of fall 2001, individual fatty acid levels were generally fairly constant. Palmitic and stearic acids have been previously reported (Samala et al., 1998; Cyril et al., 2002) to remain somewhat constant during cold acclimation. In the current study, it was found that there were variations in these fatty acids during different times of the year. However, trends were not consistent and did not hold for all cultivars.
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In the current study, N, SWE, and Fe linolenic acid levels were not different from the control even though N increased visual quality (Table 2). This suggests that these treatments did not have an effect on bermudagrass cold tolerance via a lipid-mediated mechanism.
There was a sampling date x cultivar interaction for percentage of linolenic acid to total polar lipids (Table 7). Cultivar differences in percentage of linolenic acid showed that cold-tolerant cultivars generally had higher levels than cold-sensitive cultivars. Although linolenic acid level differences were not always significant, Midiron consistently had the highest levels, followed by Riviera, Princess-77, and Tifway, respectively. Previous studies have shown that linolenic acid levels increase during cold acclimation and increase to a greater degree in cold-tolerant than cold-sensitive cultivars (Samala et al., 1998; Cyril et al., 2001, 2002). Linolenic acid levels were high in fall 2001 but dropped in winter 2002. Although it was expected that samples would have as high or higher levels of linolenic acid during this time, air temperature changes in January may at least partially explain this difference. Air temperatures leading up to the sampling date were cool, averaging around 0 to 5°C. Several days before sampling, however, the air temperature increased to 10 to 15°C and may have had a short-term effect on the degree of lipid saturation. Further, Beard (1973) explains that grasses experience their highest level of hardiness in early winter and may experience a dehardening period in mid- to late winter. Another possible explanation for high linolenic acid levels in fall 2001 is that samples had much better color retention relative to the fall 2002 sample period. Although quality was not rated in the winter months, plots of all cultivars were completely brown. A greater amount of color would equate to greater amounts of chlorophyll in bermudagrass stolons. Salisbury and Ross (1992) explain that chloroplast pigments encompass 50% of the thylakoid membrane and the fatty acid portion of this membrane has high levels of both linolenic and linoleic acid. Thus, greater amounts of stolon chloroplasts in November 2001 could have resulted in higher linolenic acid levels.
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High levels of linolenic acid did not necessarily result in greater amounts of survival following controlled freezing. Although it was found that cold-tolerant cultivars had higher levels of linolenic acid than cold-sensitive cultivars, differences within cultivars and between dates could not be correlated with survival. Riviera, Tifway, and Princess-77 all had higher levels of linolenic acid in fall 2001 than fall 2002, however, there were no differences in survival (across all temperatures) between these dates (Table 5 and 7). In winter 2003, a significant regrowth x percentage of linolenic acid correlation (r = 0.28, P = 0.01) existed at 4°C. Percentage of linolenic acid (r = 0.39, P = 0.01) correlation at –2.8°C was significant. Percentage of linolenic acid correlation at –5.0°C (r = 0.37, P = 0.01) and –7.2°C (r = 0.20, P = 0.05) were also significant. But, as can be seen, lipid unsaturation only explains 4 to 15% of regrowth. Obviously, many other factors are involved in bermudagrass cold hardiness.
The high linolenic acid levels in summer were unexpected (Table 7). As previous work in bermudagrass has shown an increase in unsaturation during the fall cold acclimation period, pre-acclimation levels were expected to be low. However, as air temperatures and light intensity during the summer were conducive to bermudagrass growth, chloroplast pigments were most likely at higher concentrations than in other times of the growing season. As was explained above, thylakoid membranes contain high levels of linolenic and linoleic acids (Salisbury and Ross, 1992). Although chloroplasts were not examined per se in the present study, they would have contributed significantly to sampled tissues mainly in the summer sampling period. The presence of the unsaturated fatty acids in the thylakoid membrane causes a higher level of fluidity (Salisbury and Ross, 1992).
Proline Concentration
Generally, proline concentration varied with cultivar (Table 8). Midiron normally had the highest levels, followed by Riviera, Tifway, and Princess-77. This finding is significant as proline concentration among cultivars follows the same trend as was shown in postfreeze regrowth (Table 5). Previous literature has examined late-season fertility affects on bermudagrass carbohydrate concentrations and has drawn conclusions on cold tolerance based on these data. There are, however, many likely physiological differences in cold-tolerant and cold-sensitive bermudagrasses including lipid unsaturation (Samala et al., 1998) and cellular proline concentration (Munshaw et al., 2004). In cold-hardiness research where proline was measured the results have indicated that cold-tolerant cultivars possess higher levels than cold-sensitive cultivars. As was shown in maize (Zea mays L.), cellular proline concentrations increase during cold acclimation (Chen and Li, 2002). As Rossi (1997) points out, an increase in cellular proline concentrations can have a large impact on osmotic adjustment during freezing events, decreasing the possibility of cytoplasm dehydration, extracellular ice formation, and cell rupture.
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Samples tested in fall 2002 showed a significant correlation between proline concentration and regrowth (r = 0.46, P = 0.01) at –7.2°C. In winter 2003, there were significant correlations between proline concentration and regrowth (r = 0.29, P = 0.01) at –2.8°C as well as at –5.0°C (r = 0.36, P = 0.01). There was also a significant correlation between proline concentration and regrowth (r = 0.38, P = 0.01) at –7.2°C.
In the present study, proline concentration was not consistently or significantly affected by Fe, SWE, or N (Table 8). There were, however, differences in terms of proline concentration between samples that were assumed to be nonacclimated (fall) and acclimated (winter). Munshaw et al. (2004) found that increasing proline concentrations in bermudagrass due to moderate salt applications was correlated with increased regrowth after freezing. Because no previous research has examined the effect of N, SWE, and Fe treatments on proline levels in bermudagrass, relationships must be drawn to the studies that have found very similar results showing no effects of these treatments on carbohydrate concentrations (White and Schmidt, 1990; Goatley et al., 1994; Richardson, 2002). Although fall quality may be affected by these chemical treatments, they appear to have no influence on physiological parameters measured in this, or other studies.
Of interest during the second season was that proline concentrations were much reduced in summer for all cultivar and chemical treatment combinations over samples taken in fall or winter (Table 8). There is likely a reduction in water uptake during periods of cool soil temperatures, resulting in a form of physiological drought. The osmotic stress that may occur during this period could result in an increase in plant proline levels. Postfreeze survival and proline concentrations showed similar trends during the summer as survival was also low for all cultivars (Table 5). It appears that high levels of lipid unsaturation are required during all times of the year in order for cellular membranes to remain fluid. However, because high levels of lipid unsaturation alone do not fully explain differences in cold tolerance, other factors such as proline concentration, TNC level, and perhaps many other physiological processes must act in concert to condition tissues for cold tolerance. This is demonstrated by findings from samples collected in July that had poor freeze tolerance (Table 5), high lipid unsaturation (Table 7), and low proline concentration (Table 8).
Spring Greenup
Percentage of greenup was rated on three dates in both 2002 and 2003. As the spring progressed, greenup increased (Table 9). Midiron and Riviera had greater amounts of greenup early in the spring and across all observation dates than Tifway and Princess-77. In 2002, Riviera and Midiron had 20 to 25% more greenup than Tifway and 55 to 75% more greenup than Princess-77 on 28 April and 15 May (Table 9). On 1 June, Riviera, Midiron, and Tifway all had higher percentage of greenup than Princess-77.
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Comparison of means within years revealed that all cultivars had higher amounts of greenup at all observation dates in 2002 than in 2003. Daily mean temperatures in the winter of 2003 were 5°C cooler on average than in 2002. Temperatures in general were cooler during the winter of 2003 and were likely a factor in greenup differences between years. The previous statement can be made with some confidence as the cold-sensitive cultivars were affected by the colder temperatures to a greater degree than the cold-tolerant cultivars and thus were much slower to greenup. Midiron showed a reduction of 36% in April 2003, while Riviera was only reduced 20%. Tifway (somewhat cold sensitive) showed a 79% reduction in greenup in 2003, and Princess-77 (cold sensitive) was reduced 98%.
In both years of the study, the cold-tolerant cultivars Midiron and Riviera rejuvenated earlier and faster than the cold-sensitive cultivars Tifway and Princess-77. Clearly, a large portion of Princess-77 was lost to winterkill in both years of the study. Munshaw and Ervin (2003) reported that, in the transition zone, Riviera had the best spring greenup of all cultivars tested in the 1997 NTEP trials. The NTEP trial also showed that Tifway had significantly worse greenup than Riviera, and Princess-77 was slower to greenup than Tifway (Morris, 2002). These results are identical to the findings in the current study as Midiron and Riviera had better greenup than Tifway and Princess-77.
There was a significant year x chemical treatment interaction (Table 10). In 2002 the June rating showed that the N treatment has significantly less greenup than the control. This response was not consistent throughout the 2002 spring rating period, nonexistent in 2003 and slight in June, thus it is likely not biologically significant. The trends of greater amounts of greenup in 2002 were consistent when considering treatments, as all were higher in 2002 than in 2003. In both years, a positive response of the treatments was not evident during greenup. Goatley et al. (1994) did not see an effect on spring greenup with late-season–applied Fe, even with rates in excess of 4 kg ha–1. White and Schmidt (1990), however, saw enhanced recovery after dormancy with fall-applied Fe at 1.2 kg ha–1. Richardson (2002), as well as Schmidt and Chalmers (1993), found that fall N applications promoted early spring greenup. In the current study, survival after controlled freezing was not affected by N treatment during either winter (Table 5).
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CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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We were unable to increase the duration of late-season growth with seaweed extract. This treatment did not result in improved turfgrass quality late in the growing season. There were also no consistent effects of SWE on postfreeze regrowth, proline concentration, lipid unsaturation, or spring greenup.
Iron treatments resulted in increased turfgrass quality over the control but only late in the growing season. This is a beneficial response, since late-season color retention and quality was a desired outcome. Late-season Fe treatments had no consistent effect on postfreeze regrowth, proline concentration, or lipid unsaturation.
Bermudagrass cultivars vary tremendously in terms of quality, recuperative capacity, and cold tolerance. Although previous research has shown differences among these cultivars in terms of cold tolerance, little physiological explanations have been offered. These results show that cultivars that are known to be cold tolerant produce higher levels of linolenic acid and proline during fall and winter months. Results from 2002 through 2003 showed significant correlations between levels of linolenic acid and proline and regrowth after freezing. This suggests the importance of enhanced levels of these compounds during the winter. However, based on the chemical treatments examined in this study, it appears that proline and lipid unsaturation are genetically controlled and are generally only affected by the environment and not by late-season inputs of N, Fe, or SWE. Cold-tolerant cultivars also showed earlier and more rapid greenup than cold-sensitive cultivars in the spring. It seems logical to conclude that cultivars less affected by stress during fall and winter break dormancy in much better physiological condition than cold-sensitive cultivars.
Recommendations for turfgrass managers in the transition zone based on data generated in this study are to use cold-tolerant cultivars such as Midiron or Riviera that exhibit good quality during the summer and fall while maximizing the likelihood of winter survival. However, because Rivera is propagated by seed, it represents an alternative establishment method for bermudagrass. Seeded cultivars can be more convienient to establish than vegetative cultivars as transportation and/or storage of vegetative planting stock is not necessary. Also, judicious N applications throughout the entire growing season along with maintaining sufficient soil K levels can prolong the growing season (Gilbert and Davis, 1971). Lengthening the period of greenness through N applications may encourage additional late-season use and improve potential revenue for golf courses and athletic fields.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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Received for publication January 25, 2005.
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSREFERENCES |
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